Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair

Abstract

Provided herein include methods and compositions for making targeted changes to a DNA sequence. In various aspects and embodiments, methods and compositions for modifying a DNA sequence in a cell (such as a plant, bacterial, yeast, fungal, algal, or mammalian cell) are provided. In some aspects and embodiments the modification of DNA involves combining gene repair oligonucleotides with approaches that enhance the availability of components of the target cell gene repair mechanisms, such as a DNA cutter.

What is claimed is: 
     
       1. A method of causing one or more targeted mutations in a flax cell, said method comprising:
 delivering a gene repair oligonucleobase (GRON) into the flax cell, wherein the GRON is a nucleic acid sequence that hybridizes at a target DNA sequence within an EPSPS gene in the flax cell genome and comprises one or more mismatched base-pair(s) relative to the target DNA sequence that encodes the targeted mutation(s), wherein the targeted mutation(s) are selected to increase glyphosate resistance in flax; and 
 culturing the flax cell under conditions that increase one or more cellular DNA repair processes prior to, coincident with, or both prior to and coincident with delivery of a GRON into the plant cell, wherein the conditions that increase one or more cellular DNA repair processes comprise introducing one or more TALENs or CRISPR nucleases into the flax cell; 
 identifying one or more flax cells comprising the targeted mutations(s) without herbicide selection by next generation sequencing of the EPSPS gene in the one or more flax cells, wherein the one or more flax cells are non-transgenic with regard to the targeted mutations(s); and 
 regenerating a flax plant from the one or more identified flax cells, wherein the flax plant is non-transgenic with regard to the targeted mutations(s) and exhibits glyphosate resistance. 
 
     
     
       2. The method of  claim 1 , wherein the GRON comprises DNA and RNA nucleotides. 
     
     
       3. The method of  claim 1 , wherein said GRON is single stranded. 
     
     
       4. The method of  claim 3 , wherein the GRON is chemically protected at the 5′ end, at the 3′ end, or at the 5′ and 3′ ends. 
     
     
       5. The method of  claim 4 , wherein the chemical protection comprises one or more moieties selected from an idC group, a Cy3 group, a group comprising three phosphorothioate internucleotide linkages, and a 2′-O-methyl group. 
     
     
       6. The method of  claim 4 , wherein the chemical protection comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the 5′ end. 
     
     
       7. The method of  claim 1 , wherein said GRON is single stranded and has a wobble base relative to the target sequence for the genetic change. 
     
     
       8. The method of  claim 1 , wherein said GRON is between 50 and 201 nucleotides in length. 
     
     
       9. The method of  claim 1  wherein the conditions that increase one or more cellular DNA repair processes comprise a CRISPR nuclease. 
     
     
       10. The method of  claim 1 , wherein the targeted mutation(s) comprise a threonine to isoleucine substitution corresponding to T1781 and a proline to alanine substitution corresponding to P182A, with amino acid numbering relative to the amino acid sequence of the Flax EPSPS protein sequence of SEQ ID NO: 12. 
     
     
       11. The method of  claim 10 , wherein the plant comprises an EPSPS gene that is homozygous for the T1781 and P182A substitutions.